Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/48—Nerve growth factor [NGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to an eisenia foetida isolated human NGF-like protein and uses thereof.
• Nerve growth factor belongs to the family of neurotrophins, growth factors playing a key role in the neuronal development in mammalians, identified only in vertebrates but thought to be essential in the evolution of quite complex nervous systems of several invertebrates, particularly invertebrate species more long-lived than well known Caenorhabditis elegans and Drosophila melanogaster (Jaaro H. et al. 2001).
• NGF consists of three subunits named ⁇ , ⁇ e ⁇ ; particularly NGF biological activity was attributed to ⁇ subunit, defined 2,5S NGF to indicate ⁇ subunits isolated from mouse salivary glands by using a method which can cause limited proteolytic processes within terminal portion of the molecule, or ⁇ -NGF or simply NGF.
• ⁇ -NGF ⁇ subunits isolated from mouse salivary glands by using a method which can cause limited proteolytic processes within terminal portion of the molecule, or ⁇ -NGF or simply NGF.
• the NGF production particularly of the growth factor ⁇ subunit, in high amounts is very interesting from the economic point of view.
• the NGF production via recombinant route in Escherichia coli bacterium is described in the patent applications EP 0121338 and US 5288622. This technique allow the production of a protein without other extract contaminating mammalian proteins which exert undesired biological activities.
• Other recombinant techniques used different expression systems like baculovirus transformed insect cells (patent application EP 037171) for the NGF expression.
• Protein production techniques via recombinant route allow, in fact, to provide NGF with high purity grade and without immunogenicity because the sequence is homologous to human one.
• these techniques suffer in that they are very expensive and laborious (long times).
• clinical experiments carried out by inoculating human recombinant NGF did not satisfy expectations resulting from NGF treatment of human pathologies in animal models (Yasuda H. et al. 2003).
• Rainworms are long considered a source of biologically active possibly useful in medical field compounds, as in the case of EFEa fibrinolytic enzyme, extracted form Eisenia foetida, able to degrade fibrin and activate the plasminogen (Tang et al., 2002).
• the authors of the present invention now identified ed isolated from Eisenia foetida a NGF-like protein which within the sequenced portion shows a 100 % homology with human NGF ⁇ subunit.
• NGF-like protein isolated from Eisenia foetida having within the amplified portion homology between 98 and 100 % with respect to ⁇ subunit of mature human nerve growth factor (NGF) in a portion corresponding to about % of molecule. Isolation of above said protein is carried out preferably from Eisenia foetida coelomocytes.
• Oligonucleotide sequences encoding for above described protein are a further object of the present invention.
• oligonucleotide sequence comprises the following encoding sequence: gtg tgg gtt ggg gat aag ace ace gcc aca gac ate aag ggc aag gag gtg atg gtg ttg gga gag gtg aac att aac aac agt gta ttc aaa cag tac ttt tt gag ace aag tgc egg gac cca aat ccc gtt gac age ggg tgc egg ggc att gac tea aag cac tgg aac tea tat tgt ace acg act cac ace ttt gtc aag gc
• oligonucleotide sequences can be desoxyribonucleotidic or ribonucleotidic or complementary sequences.
• the present invention further refers to a method for the extraction of the protein according to the invention comprising the following steps: a) isolation of coelomocytes from rainworms by treatment with extrusion solution; b) preparation of proteic extract from coelomocytes; c) protein purification by chromatography.
• Further object of the present invention is the protein achievable according to above described extraction method. It is a further object of the present invention the protein as above defined for use in medical field, particularly for the treatment of pathologies in connection with NGF defective activity.
• the protein as above defined for the preparation of a medicament for the treatment of neurodegenerative diseases of central, peripheral or autonomic nervous system like Alzheimer's disease, elderly immunodefieincy, Parkinson's disease, amiotrophic lateral sclerosis, epilepsy, Down's syndrome, nervous deafness, peripheral neuropathies, Huntington's disease, secondary neural injuries from hypoxia, ischaemia, infections (HIV, poliomyelitic virus) or trauma (including surgical ones).
• neurodegenerative diseases of central, peripheral or autonomic nervous system like Alzheimer's disease, elderly immunodefieincy, Parkinson's disease, amiotrophic lateral sclerosis, epilepsy, Down's syndrome, nervous deafness, peripheral neuropathies, Huntington's disease, secondary neural injuries from hypoxia, ischaemia, infections (HIV, poliomyelitic virus) or trauma (including surgical ones).
• the present invention refers also to the use of the protein for the preparation of a medicament for the treatment of ocular pathologies like, for example, glaucoma, retinal degeneration, etiologically different keratites (post-traumatic or post-infective conditions) and neurotrophic corneal ulcers.
• the present invention refers additionally to the use of the protein for the preparation of a medicament for the topical treatment of cutaneous ulcers both associated with human pathologies and detectable in animals and also for the preparation of a medicament for the treatment of pathologies concerning hair loss like alopecia.
• the protein according to the invention can be used for the preparation of a medicament for the treatment of pathologies wherein endothelial growth or neoangiogenesis process is to be stimulated.
• pathologies can be selected from the group consisting of ictus, aneurysms, gastrointestinal ulcers, myocardial infarction and peripheral vasculopathies.
• Further object of the present invention is the use of the protein as a factor for the promotion of growth and/or survival in vitro or ex-wVo of neural cells (as dopaminergic, cholinergic, sensorial neurons, striatal, cortical cells, as well as cells from corpus striatum, hippocampus, cerebellum, olfactive bulb, moto neurons, sympathetic neurons, neuronal staminal cells) and also for the maturation and differentiation of haematopoietic staminal cells, progenitors of immune system cells both of myeloid and lymphoid line and male reproduction system cells.
• neural cells as dopaminergic, cholinergic, sensorial neurons, striatal, cortical cells, as well as cells from corpus striatum, hippocampus, cerebellum, olfactive bulb, moto neurons, sympathetic neurons, neuronal staminal cells
• haematopoietic staminal cells progenitors of immune system cells both of myeloid and lymphoi
• an object of the present invention is a pharmaceutical composition
• a pharmaceutical composition comprising the protein object of the present invention together with pharmaceutically acceptable excipients, carriers and/or adjuvants.
• Administration of said pharmaceutical composition can be carried out via any conventional administration route, for example systemically using intravenous or parenteral injections as injectable solutions or suspensions, intramuscularly, intraparenchimally, topically or orally
• figure 1 depicts RT-PCR amplification products of conserved actin gene in Eisenia foetida coelomocytes
• figure 2 depicts primer design of a nested RT-PCR for the mature portion of NGF molecule
• figure 3 shows agarose gel electrophoresis of the external RT- PCR products
• figure 4 shows agarose gel electrophoresis of the internal RT- PCR products
• figure 5 shows the homology level between the amplified portion on retrotranscribed DNA from E. foetida total RNA and mature human NGF ( ⁇ subunit)
• EXAMPLE 1 Method for the production and extraction of NGF- iike proteins from Eisenia foetida
• animals were previously "purged” to eliminate earth grains and excrements. To this they were put in 10 cm diameter Petri dishes in number of three animals/dish and placed between two filter paper disks which together with some further added paper chips were imbibed with tap water (3 ml). Then the animals were maintained in wet environment during three days replacing the dish at the second day.
• any animal is washed with saline (NaCI, 0.85 %) at ambient temperature and then transferred in Petri dishes (3 cm diameter) containing 1.5 ml of extrusion solution.
• This solution consists of 5 % ethanol and 95 % NaCI solution containing 2.5 mg/ml of EDTA and 10 mg/ml of glyceryl ether guaiacol mucolytic agent, adjusted to pH 7 with NaOH; ethanol is added at the time of the use. Animals were maintained in the extrusion solution for two minutes and then removed.
• Cells contained in the coelomatic extruded liquid were immediately transferred in ice cooled tubes containing PBS 0.01 M or NaCI 0.85 %, both sterile and at 4°C, in a volume of 12-13 ml or 38 ml for individual or multiple samples containing coelomatic liquid from four animals, respectively. In any case finally the cells are washed thrice at 4°C by centrifugation for 5 minutes at 150xg.
• EXAMPLE 2 Study of the gene expression of NGF-like molecules in Eisenia foetida coelomocytes by RT-PCR
• I.e. coelomocytes I.e. coelomocytes.
• From any animal were obtained about 10 5 cells which were washed in cold PBS, centrifuged at 1800 rpm for 5 minutes and pellet thereof was re-suspended in 500 ⁇ l of guanidine thiocyanate (GTC) 4M solution.
• GTC is very potent denaturing agent able not only to lyse coeloma cells but also to denaturate intracellular RNases which are responsible of the rapid RNA messenger degradation.
• RNA of coelomocytes was then extracted according to the Chomczynski and Sacchi (Chomczynski P., Sacchi N, 1987) method, its integrity was verified on agarose gel electrophoresis and the obtained amount was measured by spectrophotometric reading of the optical density.
• RNAse-free DNAse-free
• MMU-LV-RT inverse transcriptase enzyme
• Primers for a nested PCR consisting of a first gene region (external) amplification and subsequent re-amplification using primers designed on a region belonging to the first (internal) were selected within the mature portion of murine ⁇ -NGF sequence ( Figure 2).
• the selection of murine NGF which shows high homology with human one, was carried out because NGF immunologically correlated proteins were detected in the present animal by the use of anti-NGF murine polyclonal antibodies which are also able to react with homologous human neurotrophins (Davoli C, et al. 2002).
• Sequenced portions corresponds to about 75 % of the human mature NGF molecule as primers used for the amplification were selected depending on the cysteines present in the mature protein which are conserved in NGF of all up to now examined species. It is noteworthy that in this portion 5 out of 6 cysteines are present and remaining one was contained in the A1 and B1 primers used for nested PCR (Table 1).
• Amplification conditions used in PCR protocol were the following: 1 ⁇ l cDNA from every sample was amplified in 20 ⁇ l final volume containing 2 mM MgCI ⁇ . 0.05 mM dNTPS and 0.5 ⁇ M primers, denaturated for 5 minutes at 95°C and 35 cycles consisting of 1 minute at
• RT-PCR products were analyzed by agarose gel electrophoresis ( Figure 4) and external PCR positive samples were re-amplified using primers for the DNA region internal to the first one.
• mice are pre-treated according to the following method: stabling of sexually mature animals at 25°C, transferring in plate and fasting in wet environment at 25°C for 3 days with plate replacing; coelomocytes collecting; washing of any animal with saline solution at ambient temperature, transferring into plate and preservation in extrusion solution (Eyambe S. G. et al., 1991) for 2 minutes, collecting of extruded cells and their transferring in sterile buffer or saline solution at 4°C with subsequent washings by centrifugation at 150xg for 5 minutes.
• Process indicated by A letter corresponds to the conventional purification method used for the preparations of NGF from mouse salivary glands with pre-treatment of the extract using streptomycin sulfate (Bocchini V., Angeletti P. U., 1969); in such a case for a better purification a further chromatographic run in CM-cellulose can be necessary (Graiani G. et al., 2004).
• Process indicated by B letter is based on chromatographic separations used for snake venom obtained NGF for which recently most appropriate purification method has been stated (Kostiza T. et al., 1995; Bian L.-J. et al., 2004).
• chromatographic separation can be by ion, cation or anion exchange.
• the fractions chromatografically isolated and SDS monitored by absorption at 280 nm are analyzed by SDS-PAGE to detect protein components while the presence of NGF-like proteins is pointed out by Western blotting, using cross-reactive polyclonal antibodies.
• Immunoreactive fractions are pooled and subjected to the following chromatography or, when sufficiently purified and dyalized, if desired, stored at -20 0 C or lyophilized and stored at 4°C.
• Biological activity is evaluated both for the ability to provoke the emission of nervous fibers by dosage in PC-12 cells or in sympathetic nervous ganglions isolated from chicken embryos (Kostiza T. et al., 1995; Bian L.-J. et al., 2004) and for the effects on immunocompetent cells (La SaIa et al., 2000).

Applications Claiming Priority (2)

Publications (1)

Family

ID=36190696

Family Applications (1)

Country Status (2)

Cited By (4)

Citations (2)

• 2005
  • 2005-02-02 IT ITRM20050045 patent/ITRM20050045A1/it unknown
• 2006
  • 2006-01-26 WO PCT/IT2006/000047 patent/WO2006082609A1/en not_active Application Discontinuation

Patent Citations (2)

Non-Patent Citations (5)

Also Published As

Similar Documents

Legal Events